1. Field of the Invention
This invention relates to improved instruments for supercritical fluid extraction of environmental, biological and other samples of materials from matrices, and for subsequent separation, clean up and concentration of the extracted samples, for example, to enrich the analytes for efficient chromatographic separation and identification. More particularly, this invention relates to an integrated instrument for performing supercritical fluid sample extraction and then separating the sample into its constituent parts, providing substantially unlimited control of the pertinent process parameters, while permitting a relatively large number of sample extraction and separation processes to be operated simultaneously and in parallel.
2. Description of the Prior Art
The science of identifying and quantifying the constituents of a sample, having been separated from one another, has advanced very substantially in the last few years. However, the process of separating typical contaminants, such as pesticides, from the matrices in which they are found, such as food products, soils and the like, has until quite recently remained relatively primitive. As stated by Hawthorne, "Analytical-Scale Supercritical Fluid Extraction", Analytical Chemistry, 62, No. 11 (1990), the sample extraction procedures employed by most laboratories have not changed since the initial reporting of chromatography in 1906, and many laboratories continue to rely on multiple distillation processes employing the so-called Soxhlet apparatus to remove the sample from the matrix.
Recently, as discussed in detail by Hawthorne, supercritical fluid extraction has been employed by more and more laboratories. A supercritical fluid is one which is above its critical temperature and pressure, such that it cannot be liquefied regardless of any increase in pressure. In this region of the state diagram of the material, it exhibits gas-like mass transfer and liquid-like solvating characteristics. That is, a supercritical fluid readily penetrates a matrix as might a gas, while materials in the matrix are dissolved by the supercritical fluid and extracted as if it were a liquid. Sample extraction using supercritical fluids is normally much faster and much simpler than repeated distillation in a Soxhlet apparatus. As supercritical fluid extraction need not be carried out at the very high temperatures characteristic of repeated distillation separation processes, thermally unstable compounds can be extracted without damage. Supercritical fluid extraction is commonly carried out using CO.sub.2 as the supercritical fluid, thus avoiding use of numerous solvents now identified as potential carcinogens such as methylene chloride, and eliminating a substantial safety hazard. Accordingly, supercritical fluid extraction of samples from matrices has become increasingly popular in recent years.
In addition to the Hawthorne article already mentioned, numerous references discuss supercritical fluid extraction. See, for example, King, "Fundamentals and Applications of Supercritical Fluid Extraction and Chromatographic Science", J. Chromatographic Sci., Vol. 27, p. 335-364 (1989); Englehardt et al, "Extraction of Pesticides from Soil with Supercritical CO.sub.2 ", J. High Resolution Chromatography and Chromatography Communications, 10772, p. 726 (1988); Katauskas et al, "SFE: Will It Solve Your Lab's Solvent Waste Problems?" R & D, March 1991, p. 39, 41-44; Parkinson et al, "Supercritical Processes Win CPI Acceptance", Chem. Eng., (July 1989), p. 35, 37, 39; Bosta et al, "Supercritical Fluids: Still Seeking Acceptance", Chem. Eng., (Feb. 4, 1985).
Other prior art references of relevance to this work suggest the use of supercritical fluids as the mobile phase in various types of chromatography. For example, Giddings et al, "Exclusion Chromatography in Dense Gases: An Approach To Viscosity Optimization", Anal. Chem., 49, No. 2 (1977) discusses the use of supercritical fluids for size exclusion chromatography of polymers in a porous silica column. Fujimoto et al, "Size Exclusion Chromatography of Polystyrenes with Supercritical Dichloromethane", J. Chromatographic Science, 27, 325, 327 (1989) discusses supercritical fluid separation of polymers by size exclusion chromatography using a porous glass matrix. However, many of the resins and other materials used for size exclusion chromatography, e.g. S-X3 resin, are not suitable for use with supercritical fluids as they degrade at the high pressures and/or relatively high temperatures required.
Other references suggest the use of particular materials such as the diatomaceous earth sold by Analytichem International as "ChemTube-Hydromatrix" for removal of moisture in supercritical fluid extraction; see Hopper et al U.S. patent application Ser. No. 07/536,861 filed Jun. 12, 1990.
Various operational parameters useful in supercritical fluid extraction are summarized by Hawthorne, supra, including the use of organic modifier solvents in addition to the supercritical fluid for extraction of relatively polar sample materials from matrices. Hawthorne also notes that the solvent strength of the supercritical fluid varies with its temperature and pressure and that these parameters can be usefully controlled for selective extraction of various samples from matrices.
The prior art also suggests combination of supercritical fluid extraction (SFE) with solid phase extraction and supercritical fluid chromatography (SFC) for on line extraction, separation, and analysis of lipid-loaded, low analyte content biological samples. See Murugaverl et al, "On-line Supercritical Extraction/Chromatography System for Trace Analysis of Pesticides in Soybean Oil and Rendered Fats", J. Microcol. Sep., 3, p. 11-16 (1991). The apparatus discussed in this reference employs a supercritical fluid extraction cell followed by a column packed with adsorbent silica or the like, and then by a fused silica capillary column for supercritical fluid chromatography (SFC). Lipid extracts are selectively retained in the packed column, and the pesticides are then concentrated by cryofocussing prior to SFC analysis. Their apparatus does not lend itself to automatic operation, as the adsorbent silica material retains high polarity substances such as organophosphate pesticides. King in the J. Chromatographic Science article supra also discusses the advantages of combining supercritical fluid extraction with selective adsorption of lipids on silica or alumina packing. Various adsorbents useful for supercritical fluid separation are summarized by Hawthorne, supra.
The art shows various commercially available instruments for supercritical fluid extraction of relatively small size (0-20 g) samples from solid or semi-solid matrices, in some cases combined with subsequent sample separation units. For example, Fisons Instruments, Valencia, Calif., offers a "Supercritical Fluid Extractor", Model SFE 30, that allows programming of the pressure of the supercritical fluid to effect selective extraction, and provides means for addition of small amounts of organic modifier to allow extraction of relatively polar analytes. The proportion of supercritical fluid to modifier can be varied through the use of two pumps.
Isco, Inc., Lincoln, Nebr., offers a Model SFX 2-10 Supercritical Fluid Extractor. This device accepts dual extraction columns but does not allow simultaneous extraction of both samples. A sample may be loaded in one column while the contents of the second column are being extracted. The Isco system provides temperature control from ambient up to 150.degree. C. Variable flow rates of supercritical fluids and modifiers are delivered by two separate refillable syringe pumps. Their outputs can be joined at a tee fitting to provide a mixture of supercritical fluid and modifier in any desired ratio.
Suprex Corporation, Pittsburgh, Pa., manufactures high pressure SFE and SFC instruments. This company offers a versatile Model MPS/225 instrument that can perform on-line SFE-SFC, SFE only, SFC only, on-line and off-line SFE followed by gas chromatography, or on-line SFE and off-line SFE followed by liquid chromatography. The Suprex device also provides for addition of solvent modifiers to the supercritical fluid.
Computer Chemical Systems, Inc., Avondale, Pa., offers a line of instruments for supercritical fluid extraction and chromatography. The Model 3200 multi-sample SFE system of this company provides for either simultaneous or sequential extraction of up to six samples. An air-driven pump is used to provide sufficient flow through all six columns. Both SFE and SFC units are microprocessor controlled, including injection valve operation, temperature and pressure. The SFC 5000 system is stated to allow unattended operation of up to sixty total sample runs using twenty sample vials. On-line mixing of supercritical fluid and an organic modifier such as methanol is also possible.
Hewlett Packard offers the HP7680A Supercritical Fluid Extractor featuring a variable restrictor nozzle allowing supercritical flow rates to be set independent of density and temperature. The system is apparently intended to be used with a variety of adsorption trap packings and can accept up to twenty-one collection vials for sample collection. This system is computer controlled using a Microsoft Windows-based system.
Newport Scientific, Jessup, Md., offers a pilot size supercritical fluid extraction system that features a large capacity diaphragm compressor capable of delivering either gas or liquid, and both large capacity SFE column and large capacity sample collection vessels. The flow direction through the extractor may be reversed by means of manually operated valves.
It can thus been seen that while the prior art has made use of supercritical fluid sample extraction combined with a wide variety of chromatographic sample separation techniques, the prior art does not show a fully automated integrated instrument providing SFE followed by supercritical fluid sample separation, including a large number of separate supercritical fluid extraction modules with independent temperature, pressure and flow rate control. Such an instrument is of great importance for laboratories processing large numbers of large and small samples, and for development of analytical methods, requiring flexibility in extraction and separation parameters.